from rat natural killer cells

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membrane sequences of four other Fc receptors including ..... TAT CAAACC AGC AATTTT TCA ATC CCC AAA GCC AAC CAC AGT CAC AGT GGCC AC TAC.

Proc. Natl. Acad. Sci. USA Vol. 87, pp. 3425-3429, May 1990 Immunology

Characterization and expression of an Fcy receptor cDNA cloned from rat natural killer cells (IgG receptor/antibody-dependent cell-mediated cytotoxicity/rat CD16)

DONNA L. ZEGER*, P. MARK HOGARTHt, AND DUANE W. SEARS* *Biochemistry and Molecular Biology Section, Department of Biological Sciences, University of California, Santa Barbara, CA 93106; and for Cancer and Transplantation, University of Melbourne, Parkville, Victoria 3052, Australia

tResearch Centre

Communicated by Ellis Englesberg, February 20, 1990 (received for review December 14, 1989)

A cDNA clone for an IgG-binding Fc recepABSTRACT tor, rtFc7,Ra, of the rat natural killer cell line CRNK-16 is characterized here. This clone encodes an FcI, receptor as shown by the ability of cDNA-transfected COS cells to rosette IgG-coated sheep erythrocytes. The rtFclRa is exceptionally homologous to the mouse moFclRa, with 77% protein sequence identity and 71% nucleic acid identity overall. The transmembrane region of the rtFc7Ra contains the sequence Leu-Phe-Ala-Val-Asp-Thr-Gly-Leu, which is present in the membrane sequences of four other Fc receptors including mouse FclRa, human FcYRHI-2, and the Fc6Ra subunits of the rat and human high-affinity IgE-binding receptors. Also, the rtFcYRa cytoplasmic domain exhibits specific homology to other receptors derived from natural killer cells, human Fc,,RIII-2 and mouse Fc,,Ra. However, the rtFclRa cDNA clone is complementary to at least two different-sized mRNAs expressed by CRNK-16 cells, contrasting the single Fc7Rrelated mRNA species expressed by human and mouse natural killer cells. These rat mRNAs are homologous to both the 5' and the 3' end of the cDNA clone, suggesting that they may be (i) splice variants of one transcript or (i) products of different but highly related genes.

The antibody Fc receptors (FcRs) expressed on the surface of immune cells form an essential functional bridge between the humoral and cellular arms of the immune system. FcRs bind the Fc domains of antibodies in antibody-antigen complexes and trigger many cell-mediated immune processes such as phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC). The primary types of receptors involved in these immune processes are the IgG-binding FcRs (FcyRs) expressed on macrophages, lymphocytes, natural killer (NK) cells, granulocytes, and a variety of other cell types. FcyRs comprise a set of structurally diverse proteins mostly encoded by a family of homologous genes. Except for one structurally dissimilar form (1), FcyRs can be classified into three groups-FcYRI, FcYRII, and FcYRIII-based on their protein sequences, ligand binding affinities, IgG subclass binding specificities, and/or cellular distributions (for reviews see refs. 2-4). Several cDNAs representing mouse and human FcYRI high-affinity receptors (5, 6), mouse and human FcYRII low-affinity receptors (7-10), and human FcYRIII low-affinity receptors (11-13) have been described and multiple cell-type-specific isoforms have been identified for each FcYR group. For example, at least three isoforms of mouse FcYRII exist-moFcRa, moFcyR,81, and moFcyR,B2-differing primarily in their transmembrane and cytoplasmic domain structures and in their expression by macrophages, lymphocytes, and other cell types (7, 14). Two isoforms of human FczRIII (CD16) have also been identified, FcYRIII-2, a transmembrane protein expressed by NK cells,

and Fc,,RIII-1, a phosphatidylinositol-glycan-linked protein expressed by neutrophils (11, 13, 15-17). The obvious complexity of FcyR structures and their regulated expression by different cell types requires a broadbased approach for defining the essential features of these molecules. Interspecies comparisons of different Fc,,R homologs have been highly informative in this regard. For example, both mouse and human FcyRIs exhibit a third highly conserved extracellular domain (5, 6) in addition to two extracellular domains characteristic of Fc,,RIIs and Fc,,RIIIs, suggesting that this third domain contributes to the unique capacity of Fc IRIs to bind monomeric antibody. While mouse and human FcyRII homologs have also been identified, species homologs of the human NK and neutrophil FcYRIII (CD16) receptors have not been clearly defined. In this study, a cDNA cloned from rat NK cells is found to encode an FcrR, designated rtFcyRa or rat CD16,4 that is exceptionally homologous to the mouse moFcRa. This receptor appears to be the functional species homolog of human huFclRIII-2 and probably mouse moFcyRa, originally identified in mouse macrophages (7) but recently shown by Northern hybridization to be homologous to the FcrR expressed by mouse NK cells (14).

MATERIALS AND METHODS Cell Lines. The following cell lines were used in this study: CRNK-16 cells, an interleukin 2-independent rat line adapted to culture (18) from a transplantable LGL (large granular lymphocyte) leukemia of Fischer 344 rats (19), generously provided by C. Reynolds (National Cancer Institute, Frederick, MD); COS cells, a simian virus 40 (SV40)-transformed monkey line (20), generously provided by C. Samuel (University of California, Santa Barbara); and J774 cells, a mouse monocytic line (21), obtained from the American Type Culture Collection. Cells were cultured in RPMI 1640 supplemented with 10 mM Hepes, 23 mM NaHCO3, 4.6 mM reduced glutathione, 0.06 mM 2-mercaptoethanol, 2 mM glutamine, 50 units ofpenicillin per ml, 50 ,ug of streptomycin sulfate per ml, and 10% heat-inactivated fetal bovine serum. For CRNK-16 cells, this medium was also supplemented with 1 mM sodium pyruvate and 1 x nonessential amino acids (Irvine Scientific). Library Screening. A rat NK-cell Agtl1 library made from CRNK-16 poly(A)+ RNA was constructed by H. Young and C. C. Yue (National Cancer Institute, Frederick, MD) (22) and generously provided by C. Reynolds. Approximately 2 X 105 plaque-forming units were screened with full-length, random-primed (23) murine FcyRf81 cDNA at 420C in 5 X SSPE/6% SDS/lOx Denhardt's solution containing herring Abbreviations: FcR, Fc receptor; NK, natural killer; SRBC, sheep red blood cell; SV40, simian virus 40. *The sequence reported in this paper has been deposited in the EMBL/GenBank data base (accession no. M32062).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

3425

3426

Immunology: Zeger et al.

sperm DNA at 0.1 mg/ml. (SSPE is 0.18 M NaCI/0.01 M NaH2PO4, pH 7.4/1 mM EDTA; Denhardt's solution is 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin.) Positive plaques were rescreened twice and one clone, A6a6, was selected for further characterization. Oligonucleotides. Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer and, after purification, they were used either as DNA sequencing primers or as probes end-labeled with [_y-32P]ATP by T4 polynucleotide kinase (24). Nucleotide Sequencing. Bacteriophage A DNA was isolated from the A6a6 clone by plate lysis (24) and the EcoRI-digested insert was subcloned into the sequencing phagemid pVZ-1, a derivative of pBluescribe (Stratagene) with an expanded polylinker site. Single-stranded DNA was isolated from the pVZ-A6a6 construct (in both orientations) by using the M13K07 helper phage (Pharmacia) and was sequenced by the dideoxy chain-termination method (25) using Sequenase (United States Biochemical). DNA sequences were analyzed and compared to other known FcR sequences with the University of Wisconsin Genetics Computer Group (UWGCG) programs (26). The 1341-base cDNA sequence of rtFcYRa was obtained from a total data base of 7189 sequenced nucleotides with an average of 5.36 independent sequencing gel determinations per nucleotide and with 90%o of the cDNA sequence read from both strands. Northern Blot Analysis. Total RNA was prepared from CRNK-16 and J774 cells by the guanidinium isothiocyanate method (24, 27). Poly(A)+ RNA was isolated by oligo(dT)-

cellulose (Collaborative Research) chromatography (28), with slight modifications (29), and was electrophoresed in a 1% agarose/0.6 M formaldehyde gel and blotted to HybondN (Amersham) as described elsewhere (6). Filters were hybridized at 60'C with random-primed rtFcYR cDNA, or with a 700-base-pair (bp) Nsi I-EcoRI 3' fragment of the cDNA (see Fig. 1), or with a 90-bp Dra I-EcoRI 3' fragment (see Fig. 1). Hybridized filters were subsequently washed in lx SSPE/0.5% SDS at 60°C and bands were visualized by autoradiography. Filters were stripped in boiling water between successive hybridizations. COS Cell Transfections and Rosetting Assay. A 1341-bp EcoRI fragment containing the entire rtFcyRa cDNA was subcloned by blunt-end ligation into the BamHI site of the SV40 expression vector pJC119 (30) (kindly provided by C. Samuel). Two pJC-A6a6 constructs, oriented in correct or incorrect orientations, were transfected into COS cells with DEAE-dextran (600 ,ug/ml; Pharmacia) (31) and chloroquine as previously described (32). Cells harvested 48 hr after transfection were tested for their ability to rosette IgG antibody-coated sheep red blood cells (SRBCs) (Sigma) prepared by preincubating a 10%o (vol/vol) suspension of SRBCs for 30 min with rabbit anti-SRBC antiserum (Cappel Laboratories) at a 1:400 dilution. IgG-coated SRBCs were added to the transfectants in phosphate-buffered saline for 15 min at 37°C, and rosetted cells were visualized by light microscopy after staining with 0.5% crystal violet. RNA was also extracted from transfectants by lysis of the cells with 0.5% Nonidet P-40 and probed for rtFcyRa-specific transcripts by dot blot analysis.

Proc. Natl. Acad. Sci. USA 87 (1990)

specific" redundant oligonucleotide probe with the sequence 5'-CTGTCACCNCTYATRKCCACGGT-3'; this sequence is anticomplementary to coding sequences for a peptide, AspSer-Gly-Glu-Tyr-Arg-Gln, found in most FcYR proteins (6). The 1.3-kb insert was subcloned into the phagemid pVZ-1 and sequenced by the strategy outlined in Fig. 1. The A6a6 clone cDNA sequence, its predicted protein sequence, and the corresponding sequences of the mouse receptor moFcYRa (7) are shown in Fig. 2. The rat-derived cDNA, designated rtFcyRa, spans 1341 nucleotides with an open reading frame of 801 nucleotides specifying a 267-amino acid protein. An N-terminal 31-amino acid signal sequence (residues -31 to -1) is predicted (33), leaving a 236-amino acid protein after cleavage. The mature rtFcyRa includes a 184-amino acid extracellular domain (residues 1-184), a 26amino acid transmembrane domain (residues 185-210), and a 26-amino acid C-terminal cytoplasmic domain (residues 211236). As indicated in Fig. 2, the extracellular domain contains five potential Asn-Xaa-Thr/Ser glycosylation sites and four cysteine residues that are most likely paired in immunoglobulinlike disulfide bonds (Cys-31 with Cys-73 and Cys-112 with Cys-156), on the basis of homology to other FcyRs and immunoglobulin superfamily protein structures (34, 35). Three additional cysteine residues exist in the transmembrane domain, but their potential disulfide-bonding status is uncertain. From the protein sequence, the calculated mass of rtFcyRa is 26.7 kDa and, after glycosylation, the mass increases to -40 kDa if one assumes an additional 2.5 kDa per site glycosylated. Comparison of Rat and Mouse FclRa. The cDNA and protein sequences of rtFcRa and moFcYRa are exceptionally homologous (Fig. 2). At the nucleotide level, 71% sequence identity is observed overall, with 75% identity in the coding regions and 43% identity in the 3' untranslated regions after insertions and gaps are introduced to optimize homology. The two protein sequences are 77% identical overall with 76% identity in the extracellular domains, 80%o identity in the transmembrane domains, and 68% identity in the cytoplasmic domains. rtFc,,Ra Expression. The full-length rtFcYRa A6a6 cDNA was subcloned into the SV40-based expression vector pJC119 and transfected into COS cells. Transient expression was assayed 48 hr later by testing the ability of transfected COS cells to rosette IgG-coated SRBCs. As shown in Fig. 3, COS cells transfected with rtFcyRa cDNA in the correct orientation relative to the SV40 promoter rosetted antibodycoated SRBCs. However, mock-transfected COS cells or cells transfected with the cDNA in the inverted orientation failed to form rosettes (data not shown). RNA dot blots of cells transfected with rtFc ,Ra in either orientation, but not of

A6a6

1

Dral RI

NsiI

RI

401

801

1201

RESULTS cDNA Isolation and Characterization. The rat CRNK-16 tumor cell line used for this study exhibits many of the phenotypic characteristics of normal rat NK cells (19). A rat Agtll cDNA library constructed from CRNK-16 poly(A)+ mRNA was screened with a full-length moFcyRI31 cDNA probe. One clone, A6a6, containing a 1.3-kilobase (kb) EcoRI insert was fully characterized and shown to be an FcYRrelated sequence by Southern blot hybridization to an "FcR-

FIG. 1. Sequencing strategy and partial restriction map of the

rtFcyRa cDNA. The 1341-bp A6a6 insert isolated from a Agtll clone

is represented in ruler-spaced increments of 20 bp. Arrows represent multiple sequencing runs performed with single- or double-stranded DNA. The EcoRI (RI) sites are derived from linkers used to construct the Agtll cDNA library.

Immunology: Zeger et al. -31 P, F

Met Thr Le. Glu Thr GCn Met Phe Gin

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Ala SCe GGy GeC CGA Trp LeA LeC PrA PrA Leu ThC MAt LeG LeuG CLe Ph Ala Phe A ACT GCA CAT TCT GGA AGC CAA TGG CTA CTC CCA CCA CTG ACA ATG TTG CTG CTG TTT GCT TTT GCA

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274 27C

Thr Trp Cly GGn Val CCn Ala Ser Tyr Thr Phe Lys Ala Thr Val Asn ACC TCCG GGC CAC GTC CAA GCC AGC TAC ACG TTT AACG GCC ACA GTC ACT -T- C--A-- -- --- --- --- --CT--- --- --- --- --- --- --- --'7 4r, Se-

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67 Asp Ser Gly Glu Tyr Arg Arg Met Ala His Thr Ser Leu Ser Asp Pro Ile His Le. Glu Val CGC AGT GGA GAA TAC CGG TGC CGA ATG GCG CAC ACC AGC CTC AGC CAC CCC ATA CAT CTC GAC GTG --- --- --- --- -CT--- --T -A- --- -A- --G --- C-- --- --- --- --TG--C-- --- -C- ---

406 40.'

89 Ile Ser Asp Trp Leu Leu Leu GSn Thr Pr. GIn Leu Val Phe Clu Cu Cly GClu Thr Ile Thr Le. ATT TCT CAC TCG CTG CTG CTC CAG ACC CCT CAA CTG GTG TTT GAG GAA GGG GMA ACC ATC ACA TTA ----- --- --- --- --- --- --- --C--G-G- --- --- CT- --- --- --- --- --- --CGC-

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AMg 111 Arg C His Ser Trp Lys Asn Lys Cln Le. Thr Lys Val Le. Leu Phe SCn Asn Cly Lys Pro Val ACC TCC CAT ACC TCC ACG AAC ACA CAC CTC ACT AMG GTC TTC TTC TTC CAC ACT CCCA AA CCT GTG --- --- --- --- --- - CG-TA --- -AC -G- A-- -CA --C --- --T--- -A --- T-C Arg A-, ACg IX S-C Ph, His (;"C SC

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155 Tyr C Lys Ala Tyr Leu Gly Arg Thr Met His Val Ser Lys Pro Val Thr Ile Thr Val GCn Gly TAC TGC AAA GCA TAT CTA GGA AGG ACA ATC CAT CTC TCC ACG CCT GTC ACC ATC ACT CTC CAA CGT --- --- --- -G- ACG- --T --- CA- --C CA- --- --- --- --- -- --- --- --- --- -ASt-

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221 Trp Arg Lys Ser Leu Ser Val Cly Lys Tyr Lys Ala Pro Gln Asp Lys Tr. 237 CyC CCC CAA CCC CCC ACC GA TC .CCCATCGTATGG TCC AGG AAA TCCCCTG TCC GyTC 86C TCG ------- A-- A-- G-------T C-A ----- C-- C-- ---G --- -- C-CCAT ----- C- ---- 868 Iie Arg Hl~s (;ir !, Tr,"l

CTCCACA GCGGCACCTTCTTTTCACCCCACCCCCCCCTTCATCTACACCCTTCCTTTGAGCCACTTACCACCACG CTCCATAT 9C 3 -A-----TA--ATGTT--CG-GG----AG--A-TTTT-A-CAC---------C---------------A-C--- C-C---C 954

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Proc. Natl. Acad. Sci. USA 87 (1990)

:21 19, C: 7-.

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mock-transfected cells, revealed high expression levels of rtFc.Ra RNA (data not shown). As shown in Fig. 4A, Northern blot analysis of poly(A)+ RNA isolated from CRNK-16 and J774 cells probed with the full-length rtFc.Ra cDNA clone revealed at least two CRNK16 transcripts, 1.6 and 1.4 kb in size, and a broad crosshybridizing J774 band; the width of the J774 band encompasses the range of sizes found for the three highly homologous FcyR isoforms-moFcYR,81 (1.6 kb), moFcYR,82 (1.5 kb) and moFcyRa (1.4 kb)-known to be expressed by this cell line (ref. 7 and D.W.S., unpublished data). A small (

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